scholarly journals Association of β-Arrestin 1 with the Type 1A Angiotensin II Receptor Involves Phosphorylation of the Receptor Carboxyl Terminus and Correlates with Receptor Internalization

2001 ◽  
Vol 15 (10) ◽  
pp. 1706-1719
Author(s):  
Hongwei Qian ◽  
Luisa Pipolo ◽  
Walter G. Thomas
Keyword(s):  
Angiotensin Ii ◽  
Close Correlation ◽  
Dominant Negative ◽  
Receptor Internalization ◽  
Central Portion ◽  
Carboxyl Terminus ◽  
Angiotensin Ii Receptor ◽  
Receptor Phosphorylation ◽  
At1a Receptor ◽  
Mutant Receptors

Abstract Arrestins bind to phosphorylated G protein-coupled receptors and participate in receptor desensitization and endocytosis. Although arrestins traffic with activated type 1 (AT1A) angiotensin II (AngII) receptors, the contribution of arrestins to AT1A receptor internalization is controversial, and the physical association of arrestins with the AT1A receptor has not been established. In this study, by coimmunoprecipitating AT1A receptors and β-arrestin 1, we provide direct evidence for an association between arrestins and the AT1A receptor that was agonist- and time-dependent and contingent upon the level ofβ -arrestin 1 expression. Serial truncation of the receptor carboxyl terminus resulted in a graded loss of β-arrestin 1 association, which correlated with decreases in receptor phosphorylation. Truncation of the AT1A receptor to lysine325 prevented AngII-induced phosphorylation and β-arrestin 1 association as well as markedly inhibiting receptor internalization, indicating a close correlation between these receptor parameters. AngII-induced association was also dramatically reduced in a phosphorylation- and internalization-impaired receptor mutant in which four serine and threonine residues in the central portion of the AT1A receptor carboxyl terminus (Thr332, Ser335, Thr336, Ser338) were substituted with alanine. In contrast, substitutions in another serine/threonine-rich region (Ser346, Ser347, Ser348) and at three PKC phosphorylation sites (Ser331, Ser338, Ser348) had no effect on AngII-inducedβ -arrestin 1 association or receptor internalization. While AT1A receptor internalization could be inhibited by a dominant-negative β-arrestin 1 mutant (βarr1319–418), treatment with hyperosmotic sucrose to inhibit internalization did not abrogate the differences in arrestin association observed between the wild-type and mutant receptors, indicating that arrestin binding precedes, and is not dependent upon, receptor internalization. Interestingly, a substituted analog of AngII,[ Sar1Ile4Ile8]-AngII, which promotes robust phosphorylation of the receptor but does not activate receptor signaling, stimulated strong β-arrestin 1 association with the full-length AT1A receptor. These results identify the central portion of the AT1A receptor carboxyl terminus as the important determinant for β-arrestin 1 binding and internalization and indicate that AT1A receptor phosphorylation is crucial for β-arrestin docking.

scholarly journals Identification of protein kinase C phosphorylation sites in the angiotensin II (AT1A) receptor

1999 ◽  
Vol 343 (3) ◽  
pp. 637-644 ◽  
Author(s):  
Hongwei QIAN ◽  
Luisa PIPOLO ◽  
Walter G. THOMAS
Keyword(s):  
Protein Kinase C ◽  
Angiotensin Ii ◽  
Protein Kinase ◽  
Single Mutation ◽  
Wild Type ◽  
Receptor Phosphorylation ◽  
Kinase C ◽  
C Terminus ◽  
At1a Receptor ◽  
Mutant Receptors

Protein kinase C (PKC) phosphorylates the C-terminus of the type 1 angiotensin II receptor (AT1), although the exact site(s) of phosphorylation are unidentified. In the present study, we examined the phosphorylation of epitope-tagged wild-type AT1A receptors, transiently expressed in Chinese hamster ovary K1 cells, in response to angiotensin II (AngII) and following selective activation and inhibition of PKC. This phosphorylation was compared with mutant receptors where C-terminal serine residues (Ser331, Ser338 and Ser348) within three putative PKC consensus sites were replaced with alanine, either individually or in combination. Stimulation by AngII or the phorbol ester PMA to activate PKC induced an increase in phosphorylation of the wild-type AT1A receptor, which was prevented by truncation of the receptor C-terminus to remove the last 34 amino acids, including Ser331, Ser338 and Ser348. Whereas single alanine mutation (Ser331Ala, Ser338Ala and Ser348Ala) resulted in decreased receptor phosphorylation, no single mutant completely inhibited either AngII- or PMA-induced phosphorylation. Combined mutation of the three PKC consensus sites caused an ≈ 70% reduction in PMA-mediated phosphorylation. The ≈ 60% reduction in AngII (1 μM)-induced phosphorylation of this triple mutant and the partial inhibition of wild-type receptor phosphorylation by bisindolylmaleimide, a specific PKC inhibitor, suggest a significant contribution of PKC to agonist-stimulated regulation. The ratio of PKC to total receptor phosphorylation was greatest at low doses of AngII (1 nM), consistent with the idea that PKC phosphorylates and regulates receptor function at low levels of stimulation, whereas phosphorylation by other kinases is more prevalent at high levels of agonist stimulation. To determine if a single PKC site is favoured when the contribution of PKC varies, the phosphorylation of wild-type and mutant receptors was examined over a range of AngII concentrations (0, 1, 10 and 100 nM). At all AngII concentrations, single mutation of Ser331, Ser338 or Ser348 was incapable of completely preventing receptor phosphorylation, suggesting no clear preference for PKC consensus-site utilization. Together, these results indicate a redundancy in PKC phosphorylation of the AT1A receptor, whereby all three consensus sites are utilized to some degree following homologous (AngII) and heterologous (PMA) stimulation. The contribution of PKC phosphorylation to receptor regulation is unclear, but multiple PKC phosphorylation of the AT1A receptor may allow independent and/or complementary events to occur at the three separate sites of the C-terminus.

scholarly journals Phospholipase D2 Localizes to the Plasma Membrane and Regulates Angiotensin II Receptor Endocytosis

2004 ◽  
Vol 15 (3) ◽  
pp. 1024-1030 ◽  
Author(s):  
Guangwei Du ◽  
Ping Huang ◽  
Bruce T. Liang ◽  
Michael A. Frohman
Keyword(s):  
Plasma Membrane ◽  
Angiotensin Ii ◽  
Membrane Vesicle ◽  
Membrane Vesicles ◽  
Cell Types ◽  
Dominant Negative ◽  
Secretory Cells ◽  
Resting Cells ◽  
Angiotensin Ii Receptor ◽  
Multiple Cell

Phospholipase D (PLD) is a key facilitator of multiple types of membrane vesicle trafficking events. Two PLD isoforms, PLD1 and PLD2, exist in mammals. Initial studies based on overexpression studies suggested that in resting cells, human PLD1 localized primarily to the Golgi and perinuclear vesicles in multiple cell types. In contrast, overexpressed mouse PLD2 was observed to localize primarily to the plasma membrane, although internalization on membrane vesicles was observed subsequent to serum stimulation. A recent report has suggested that the assignment of PLD2 to the plasma membrane is in error, because the endogenous isoform in rat secretory cells was imaged and found to be present primarily in the Golgi apparatus. We have reexamined this issue by using a monoclonal antibody specific for mouse PLD2, and find, as reported initially using overexpression studies, that endogenous mouse PLD2 is detected most readily at the plasma membrane in multiple cell types. In addition, we report that mouse, rat, and human PLD2 when overexpressed all similarly localize to the plasma membrane in cell lines from all three species. Finally, studies conducted using overexpression of wild-type active or dominant-negative isoforms of PLD2 and RNA interference-mediated targeting of PLD2 suggest that PLD2 functions at the plasma membrane to facilitate endocytosis of the angiotensin II type 1 receptor.

Angiotensin II receptor internalization and signaling in isolated rat hepatocytes

1999 ◽  
Vol 57 (10) ◽  
pp. 1125-1131 ◽  
Author(s):  
Eugenio Jiménez ◽  
Maria C Caro ◽  
Santo Marsigliante ◽  
Mercedes Montiel
Keyword(s):  
Angiotensin Ii ◽  
Rat Hepatocytes ◽  
Receptor Internalization ◽  
Angiotensin Ii Receptor ◽  
Isolated Rat Hepatocytes ◽  
Isolated Rat

scholarly journals Phosphorylation of the Angiotensin II (AT1A) Receptor Carboxyl Terminus: A Role in Receptor Endocytosis

1998 ◽  
Vol 12 (10) ◽  
pp. 1513-1524 ◽  
Author(s):  
Walter G. Thomas ◽  
Thomas J. Motel ◽  
Christopher E. Kule ◽  
Vijay Karoor ◽  
Kenneth M. Baker
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Angiotensin Ii ◽  
Chinese Hamster ◽  
Carboxyl Terminus ◽  
Amino Acid Substitutions ◽  
Ang Ii ◽  
Acidic Amino Acid ◽  
Wild Type ◽  
At1a Receptor

Abstract The molecular mechanism of angiotensin II type I receptor (AT1) endocytosis is obscure, although the identification of an important serine/threonine rich region (Thr332Lys333Met334Ser335Thr336Leu337Ser338) within the carboxyl terminus of the AT1A receptor subtype suggests that phosphorylation may be involved. In this study, we examined the phosphorylation and internalization of full-length AT1A receptors and compared this to receptors with truncations and mutations of the carboxyl terminus. Epitope-tagged full-length AT1A receptors, when transiently transfected in Chinese hamster ovary (CHO)-K1 cells, displayed a basal level of phosphorylation that was significantly enhanced by angiotensin II (Ang II) stimulation. Phosphorylation of AT1A receptors was progressively reduced by serial truncation of the carboxyl terminus, and truncation to Lys325, which removed the last 34 amino acids, almost completely inhibited Ang II-stimulated 32P incorporation into the AT1A receptor. To investigate the correlation between receptor phosphorylation and endocytosis, an epitope-tagged mutant receptor was produced, in which the carboxyl-terminal residues, Thr332, Ser335, Thr336, and Ser338, previously identified as important for receptor internalization, were substituted with alanine. Compared with the wild-type receptor, this mutant displayed a clear reduction in Ang II-stimulated phosphorylation. Such a correlation was further strengthened by the novel observation that the Ang II peptide antagonist, Sar1Ile8-Ang II, which paradoxically causes internalization of wild-type AT1A receptors, also promoted their phosphorylation. In an attempt to directly relate phosphorylation of the carboxyl terminus to endocytosis, the internalization kinetics of wild-type AT1A receptors and receptors mutated within the Thr332-Ser338 region were compared. The four putative phosphorylation sites (Thr332, Ser335, Thr336, and Ser338) were substituted with either neutral [alanine (A)] or acidic amino acids [glutamic acid (E) and aspartic acid (D)], the former to prevent phosphorylation and the latter to reproduce the acidic charge created by phosphorylation. Wild-type AT1A receptors, expressed in Chinese hamster ovary cells, rapidly internalized after Ang II stimulation [t1/2 2.3 min; maximal level of internalization (Ymax) 78.2%], as did mutant receptors carrying single acidic substitutions (T332E, t1/2 2.7 min, Ymax 76.3%; S335D, t1/2 2.4 min, Ymax 76.7%; T336E, t1/2 2.5 min, Ymax 78.2%; S338D, t1/2 2.6 min, Ymax 78.4%). While acidic amino acid substitutions may simply be not as structurally disruptive as alanine mutations, we interpret the tolerance of a negative charge in this region as suggestive that phosphorylation may permit maximal internalization. Substitution of all four residues to alanine produced a receptor with markedly reduced internalization kinetics (T332A/S335A/T336A/S338A, t1/2 10.1 min, Ymax 47.9%), while endocytosis was significantly rescued in the corresponding quadruple acidic mutant (T332E/S335D/T336E/S338D, t1/2 6.4 min, Ymax 53.4%). Double mutation of S335 and T336 to alanine also diminished the rate and extent of endocytosis (S335A/T336A, 3.9 min, Ymax 69.3%), while the analogous double acidic mutant displayed wild type-like endocytotic parameters (S335D/T336E, t1/2 2.6 min, Ymax 77.5%). Based on the apparent rescue of internalization by acidic amino acid substitutions in a region that we have identified as a site of Ang II-induced phosphorylation, we conclude that maximal endocytosis of the AT1A receptor requires phosphorylation within this serine/threonine-rich segment of the carboxyl terminus.

scholarly journals Carboxyl-terminal and intracellular loop sites for CRF1 receptor phosphorylation and β-arrestin-2 recruitment: a mechanism regulating stress and anxiety responses

2007 ◽  
Vol 293 (1) ◽  
pp. R209-R222 ◽  
Author(s):  
Robert H. Oakley ◽  
J. Alberto Olivares-Reyes ◽  
Christine C. Hudson ◽  
Fabiola Flores-Vega ◽  
Frank M. Dautzenberg ◽  
...  
Keyword(s):  
Cell Membrane ◽  
Receptor Internalization ◽  
Carboxyl Terminus ◽  
Receptor Interaction ◽  
Intracellular Loop ◽  
Membrane Expression ◽  
Receptor Phosphorylation ◽  
Third Intracellular Loop ◽  
Carboxyl Terminal ◽  
Factor Type

The primary goal was to test the hypothesis that agonist-induced corticotropin-releasing factor type 1 (CRF1) receptor phosphorylation is required for β-arrestins to translocate from cytosol to the cell membrane. We also sought to determine the relative importance to β-arrestin recruitment of motifs in the CRF1 receptor carboxyl terminus and third intracellular loop. β-Arrestin-2 translocated significantly more rapidly than β-arrestin-1 to agonist-activated membrane CRF1 receptors in multiple cell lines. Although CRF1 receptors internalized with agonist treatment, neither arrestin isoform trafficked with the receptor inside the cell, indicating that CRF1 receptor-arrestin complexes dissociate at or near the cell membrane. Both arrestin and clathrin-dependent mechanisms were involved in CRF1 receptor internalization. To investigate molecular determinants mediating the robust β-arrestin-2-CRF1 receptor interaction, mutagenesis was performed to remove potential G protein-coupled receptor kinase phosphorylation sites. Truncating the CRF1 receptor carboxyl terminus at serine-386 greatly reduced agonist-dependent phosphorylation but only partially impaired β-arrestin-2 recruitment. Removal of a serine/threonine cluster in the third intracellular loop also significantly reduced CRF1 receptor phosphorylation but did not alter β-arrestin-2 recruitment. Phosphorylation was abolished in a CRF1 receptor possessing both mutations. Surprisingly, this mutant still recruited β-arrestin-2. These mutations did not alter membrane expression or cAMP signaling of CRF1 receptors. Our data reveal the involvement of at least the following two distinct receptor regions in β-arrestin-2 recruitment: 1) a carboxyl-terminal motif in which serine/threonine residues must be phosphorylated and 2) an intracellular loop motif configured by agonist-induced changes in CRF1 receptor conformation. Deficient β-arrestin-2-CRF1 receptor interactions could contribute to the pathophysiology of affective disorders by inducing excessive CRF1 receptor signaling.

Electrostatic and Hydrophobic Forces Tether the Proximal Region of the Angiotensin II Receptor (AT1A) Carboxyl Terminus to Anionic Lipids†

Biochemistry ◽  
2002 ◽  
Vol 41 (24) ◽  
pp. 7830-7840 ◽  
Author(s):  
Henriette Mozsolits ◽  
Sharon Unabia ◽  
Ariani Ahmad ◽  
Craig J. Morton ◽  
Walter G. Thomas ◽  
...  
Keyword(s):  
Angiotensin Ii ◽  
Proximal Region ◽  
Carboxyl Terminus ◽  
Angiotensin Ii Receptor ◽  
Anionic Lipids
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